Stuff you need:
10X running buffer:
50 mM NaOAc
0.2M MOPS pH 7.0
10 mM EDTA
filter, store in dark at RT, pH to 7.0
0.9% agarose in 1X running buffer, and 1ml formaldehyde per 20 ml gel. (For 200
ml gel, dissolve 1.8 g agarose in 170 ml water, let cool in 65 deg C water bath,
add 10 ml formaldehyde (37% stock), and 20 ml 10X MOPS running buffer).
1.5X Loading buffer:
750 µl formamide
75 µl formaldehyde (37%)
150 µl 10X running buffer
15 µl EtBr (5 mg/ml)
10 µl ddH20
Best results if made fresh each time
Bromophenol blue in 35% glycerol and 1X running buffer
0.3M Na Citrate
- Pour gel in hood.
- Mix RNA (10µg) with 1.5X loading buffer (e.g. 10 µl RNA + 20µl LB).
- Incubate 5 min at 65 deg C. Cool on ice.
- Add 2 µl loading dye.
- Pre-run gel at 5 V/cm for 5 minutes. Load samples and run. Mix buffers after
- Wash gel 2X in water for 15 min, then 1X in 10X SSC for 15 minutes. Photograph
gel under UV.
- Cut nylon membrane (MSI 0.45 micron #N04HY00010) and several pieces of blotting
(e.g. Schleicher and Schuell GB002) paper to the same size as the gel. Wet the
nylon with dH20, then soak in 5x SSC.
- Assemble sandwich:
- Large sheet of plastic wrap
- Two pieces blot paper (precut to same size as gel) soaked in 20x SSC
- Gel (wells-side down)
- Presoaked nylon
- One piece blot paper soaked in 5x SSC
- 10-15 pieces of dry blot paper
- Wrap whole sandwich in the plastic wrap
- Place glass plate and weight on top and let transfer >3 hr.
- Crosslink after blotting.
- Can probe either with non-radioactive probe or
with radioactive probe.